Our recent studies have shown that the effective treatment of chronic myeloid leukemia (CML) with donor lymphocyte infusion (DLI) induces a polyclonal immune response targeting many CML-associated antigens. While DLI is one of the most effective immune-based therapies for cancer, it requires a matched donor and is preceded by bone marrow transplant. To overcome these limitations, we are developing a universal CML immunotherapy that may also stimulate an effective polyclonal anti-CML response. Recent trials at our institution and others have shown that anti-tumor immunity is possible to induce in patients immunized with autologous irradiated tumor cells engineered to express GM-CSF via adenoviral infection. Challenges of this approach include harvesting sufficient quantities of tumor cells for vaccination, effectively infecting cells with GM-CSF-expressing virus and generating consistent GM-CSF secretion. Variations in these parameters make it difficult to optimize any protocol using this approach. To address these problems in CML, in collaboration with the Harvard Gene Therapy Laboratory, we have recently engineered a stable K562 cell line (CML-derived) to secrete higher and more consistent levels of GM-CSF than previously achievable. This new cell line, designated GM-K562, can be used as a bystander line to co-inject with irradiated autologous tumor cells, or directly as a CML-specific vaccine as it naturally expresses CML-associated antigens. In Aim 1, we describe the primary objective of the trial, which is to evaluate safety and toxicity of GM-K562 vaccination, a toxicity monitoring plan, the expected outcome and stopping rules. As we anticipate the toxicity to be low based on previous GM-CSF trials, we propose in Aim 2 to assess the efficacy of vaccination using a sensitive PCR-based approach to measure tumor burden. Based on our experience in monitoring anti-tumor immunity in DLI-treated CML patients, we describe in Aim 3 approaches to characterize immune cell recruitment to the site of vaccination and to quantitate anti-tumor immunity by B and T cells. It should be possible to detect immunity in these patients as they are immuno-competent and can attain a stable minimal residual disease status following imatinib monotherapy. The GM-K562 cell line is thus a universal and reproducible vaccination reagent that provides a unique opportunity to a) test a novel immunotherapy reagent for CML; b) test a promising reagent for bystander vaccination with other tumors and c) generate quantitative dose-response data that will help design any future GM-CSF-based trials. The goal of this proposal is to carry out an initial phase I trial using this potentially valuable reagent and thus provide a basis for the continuation and refinement of clinical trials using this reagent.